Hepatitis B virus (HBV) remains a major worldwide public health challenge with 10-20 million chronically infected individuals developing cirrhosis, characterized by a decline and loss of liver function with a significantly decreased quality of life and a high mortality rate. While current therapies effectively control the virus with few side effects, they do not cure since none target the HBV covalently closed- circular DNA (cccDNA; associated with viral persistence). Our innovative approach targets capsid assembly which is essential for replication, as DNA synthesis from cccDNA occurs exclusively within the capsid encoded particle. Therefore, the focus of this proposal is on developing small molecules targeting disruption of capsid formation which could ultimately impact cccDNA stability and/or formation and also reduce duration of treatment in HBV infected persons. We are developing a novel class of Capsid Assembly Effectors (CAE) that target capsid formation (glyoxamide derivatives). Recently, we have identified a highly potent ?lead? compound IV with selective activity at 3 nM. The proposed specific AIMS will systematically define the pre-clinical parameters (including activity, toxicity, stability, oral bioavailability, capsid binding interaction) of this novel class of CAE: AIM 1: to chemically optimize and characterize a unique series of glyoxamide CAE, including our ?lead? compound IV; AIM 2: to structurally, biochemically, and biologically characterize novel CAE binding interaction with HBV capsid.; and AIM 3: to determine pharmacokinetics (PK) and efficacy of novel CAE and combination regimens in two different mouse models for HBV infection. Results from these studies will validate our novel class of small molecule CAE, which when combined with other modalities (e.g., nucleoside analogs) could provide preclinical ?proof of concept? towards a novel therapeutic strategy with reduced treatment duration and a functional cure for HBV infection.